环境智能响应纳米瘤苗联合光动力疗法精准靶向胃癌干细胞的研究

Gastric cancer is one of the most common malignant tumors in the world. It is well known as a serious threat to human health. Targeted killing CSCs is a new direction of tumor therapy in recent years. A novel self-degradable polymer is used as the basic material to synthesize environment-responsive nanocarrier. The nanocarrier is coupled with specific marker LGR5 of gastric CSCs and immune adjuvant CpG by electrostatic assembly and hydrophobic interaction method. Thus, we construct smart environment-responsive nanoparticles vaccine (p@a/c@SNCs). Our research is carried out by several modes, including cell mode in vitro, two models of mice bearing mice gastric LGR5 positive CSCs subcutaneously transplanted tumor and immune reconstructed SCID mice bearing human gastric LGR5 positive CSCs orthotopic transplanted tumor in vivo. The p@a/c@SNCs vaccine is locally aggregated in the tumor and environmental response to activate the self-degradation for the controlled release of CpG and LGR5 presented by DCs to activate the targeted immune response of specific CTLs to gastric LGR5 positive CSCs. At the same time, we make the NIR illumination on the tumor area. The photosensitizer carried by this nanoparticle vaccine is activated to generate ROS to make the photodynamic therapy for gastric LGR5 positive CSCs. This photodynamic therapy causes a rapid death of CSCs and produces tumor antigens in situ to strengthen the anti-tumor immune response. In a word, the smart environment-responsive p@a/c@SNCs nanoparticle vaccine is constructed in our research to make a novel strategy of coordinating targeted immunotherapy and precise photodynamic therapy together to make a new therapeutic strategy for gastric cancer.

胃癌是全球常见的恶性肿瘤，严重威胁人类健康。靶向杀伤肿瘤干细胞（CSCs）是肿瘤治疗的新方向。本研究在新型自降解聚合物的基础上，通过静电组装和疏水作用嵌合胃癌CSCs特异标志物LGR5和免疫佐剂CpG，制备环境智能响应纳米瘤苗（p@a/c@SNCs）。通过细胞模型、胃癌LGR5阳性CSCs的鼠源皮下移植瘤模型以及SCID小鼠免疫重建人源原位移植瘤模型，研究p@a/c@SNCs纳米瘤苗在肿瘤局部富集、响应环境释放LGR5和CpG、经DCs呈递激活CTLs靶向胃癌LGR5阳性CSCs的特异性免疫应答，以及在肿瘤局部经NIR激发纳米瘤苗所携带光敏剂，产生ROS进行光动力治疗，导致胃癌LGR5阳性CSCs坏死并原位产生肿瘤抗原，强化免疫应答的系列效应。本项目通过使用p@a/c@SNCs智能纳米瘤苗，以实现靶向免疫治疗与精准光动力疗法相结合的胃癌新型治疗策略。

胃癌是全球常见恶性肿瘤，严重威胁人类健康。肿瘤干细胞(CSCs)是引起肿瘤耐药、复发和预后不良的重要因素，靶向杀伤CSCs是肿瘤治疗的新方向。本研究收集胃癌患者的临床标本，采用免疫组化检测LGR5、CD24和CD44的表达，研究胃癌干细胞特征，并利用临床数据进行预后相关分析。通过无血清培养筛选胃癌类CSCs，使用Western-Blot检测LGR5和Wnt/β-catenin通路相关性以及LGR5参与肿瘤干性维持和EMT的分子机制。并且通过纳米生物工程材料和医学的学科交叉，模拟自然界蛋白质分子之间的相互作用，自主合成可降解仿生聚合物纳米载体。进一步通过静电组装和疏水作用，嵌合LGR5蛋白和DADS，制备环境智能响应纳米药物（p@a/d@SNCs）。针对光动力疗法（PDT）的特点，使其在肿瘤微环境局部pH/光/H2O2/GSH不同差异条件刺激下，基于时空精准控制，在肿瘤局部发生自降解进而释放LGR5蛋白和DADS，同时联合PDT实现更加有效的抗肿瘤疗效。研究结果显示，LGR5+细胞簇呈CD44+/CD24-性质，LGR5为胃癌CSCs的生物标志并且与患者不良预后相关。LGR5可激活Wnt/β-catenin通路，与TGF-β通路发生串联以维持肿瘤干性并且促进EMT。因此，LGR5可以成为胃癌新的诊断和治疗靶点。此外，研究证实p@SNCs纳米药物具有较强的溶解度、良好的靶向性以及可操控性，是理想的药物载体。本研究成功合成负载LGR5/DADS的p@a/d@SNCs纳米药物。研究证实，该纳米药物能够响应肿瘤局部光/pH/H2O2/GSH环境差异刺激，自降解促进药物释放和吸收。同时联合PDT疗法，成功实现时空精准控制下的药物释放，在肿瘤局部定向靶向胃癌LGR5+CSCs细胞，有效抑制其增殖并诱导其凋亡。本研究为未来p@ a/d@SNCs转化于临床应用奠定基础，提供了一种基于时空精准控制下使用智能响应纳米药物治疗胃癌的新策略。